Z-nucleic-acid sensing triggers ZBP1-dependent necroptosis and inflammation

Abstract

The biological function of Z-DNA and Z-RNA, nucleic acid structures with a left-handed double helix, is poorly understood1,2,3. Z-DNA-binding protein 1 (ZBP1; also known as DAI or DLM-1) is a nucleic acid sensor that contains two Zα domains that bind Z-DNA4,5 and Z-RNA6,7,8. ZBP1 mediates host defence against some viruses6,7,9,10,11,12,13,14 by sensing viral nucleic acids6,7,10. RIPK1 deficiency, or mutation of its RIP homotypic interaction motif (RHIM), triggers ZBP1-dependent necroptosis and inflammation in mice15,16. However, the mechanisms that induce ZBP1 activation in the absence of viral infection remain unknown. Here we show that Zα-dependent sensing of endogenous ligands induces ZBP1-mediated perinatal lethality in mice expressing RIPK1 with mutated RHIM (Ripk1mR/mR), skin inflammation in mice with epidermis-specific RIPK1 deficiency (RIPK1E-KO) and colitis in mice with intestinal epithelial-specific FADD deficiency (FADDIEC-KO). Consistently, functional Zα domains were required for ZBP1-induced necroptosis in fibroblasts that were treated with caspase inhibitors or express RIPK1 with mutated RHIM. Inhibition of nuclear export triggered the Zα-dependent activation of RIPK3 in the nucleus resulting in cell death, which suggests that ZBP1 may recognize nuclear Z-form nucleic acids. We found that ZBP1 constitutively bound cellular double-stranded RNA in a Zα-dependent manner. Complementary reads derived from endogenous retroelements were detected in epidermal RNA, which suggests that double-stranded RNA derived from these retroelements may act as a Zα-domain ligand that triggers the activation of ZBP1. Collectively, our results provide evidence that the sensing of endogenous Z-form nucleic acids by ZBP1 triggers RIPK3-dependent necroptosis and inflammation, which could underlie the development of chronic inflammatory conditions—particularly in individuals with mutations in RIPK1 and CASP817,18,19,20.

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Fig. 1: ZBP1 causes skin inflammation in RIPK1E-KO mice and perinatal lethality in Ripk1mR/mR mice by Zα-dependent and -independent mechanisms.
Fig. 2: Zα-dependent sensing of Z-NAs induces ZBP1-mediated necroptosis and colitis when FADD–caspase-8 function is inhibited.
Fig. 3: The Zα domains of ZBP1 bind cellular dsRNA probably derived from EREs.
Fig. 4: Inhibition of nuclear export triggers Zα-dependent ZBP1-mediated cell death.

Data availability

The original RNA-sequencing data are uploaded and available at the Gene Expression Omnibus (GEO) under accession GSE143955. Source Data for Figs. 14 and Extended Data Figs. 26, 8, 9 are provided with the paper.

Code availability

A GTF file of repetitive region annotations for the mouse genome (GRCm38.78), with the adjacent annotations for the same element merged is included in Supplementary Data 1. Specific shell commands executed and Python code to convert k-mers into complementary k-mers are included in Supplementary Data 2.

Change history

  • 03 April 2020

    An amendment to this paper has been published and can be accessed via a link at the top of the paper.

References

  1. 1.

    Wang, A. H. et al. Molecular structure of a left-handed double helical DNA fragment at atomic resolution. Nature 282, 680–686 (1979).

  2. 2.

    Rich, A. & Zhang, S. Z-DNA: the long road to biological function. Nat. Rev. Genet. 4, 566–572 (2003).

  3. 3.

    Herbert, A. Z-DNA and Z-RNA in human disease. Commun. Biol. 2, 7 (2019).

  4. 4.

    Ha, S. C. et al. The crystal structure of the second Z-DNA binding domain of human DAI (ZBP1) in complex with Z-DNA reveals an unusual binding mode to Z-DNA. Proc. Natl Acad. Sci. USA 105, 20671–20676 (2008).

  5. 5.

    Schwartz, T., Behlke, J., Lowenhaupt, K., Heinemann, U. & Rich, A. Structure of the DLM-1–Z-DNA complex reveals a conserved family of Z-DNA-binding proteins. Nat. Struct. Biol. 8, 761–765 (2001).

  6. 6.

    Maelfait, J. et al. Sensing of viral and endogenous RNA by ZBP1/DAI induces necroptosis. EMBO J. 36, 2529–2543 (2017).

  7. 7.

    Thapa, R. J. et al. DAI senses influenza a virus genomic RNA and activates RIPK3-dependent cell death. Cell Host Microbe 20, 674–681 (2016).

  8. 8.

    Placido, D., Brown, B. A., II, Lowenhaupt, K., Rich, A. & Athanasiadis, A. A left-handed RNA double helix bound by the Zα domain of the RNA-editing enzyme ADAR1. Structure 15, 395–404 (2007).

  9. 9.

    Guo, H. et al. Species-independent contribution of ZBP1/DAI/DLM-1-triggered necroptosis in host defense against HSV1. Cell Death Dis. 9, 816 (2018).

  10. 10.

    Sridharan, H. et al. Murine cytomegalovirus IE3-dependent transcription is required for DAI/ZBP1-mediated necroptosis. EMBO Rep. 18, 1429–1441 (2017).

  11. 11.

    Koehler, H. et al. Inhibition of DAI-dependent necroptosis by the Z-DNA binding domain of the vaccinia virus innate immune evasion protein, E3. Proc. Natl Acad. Sci. USA 114, 11506–11511 (2017).

  12. 12.

    Kuriakose, T. et al. ZBP1/DAI is an innate sensor of influenza virus triggering the NLRP3 inflammasome and programmed cell death pathways. Sci. Immunol. 1, aag2045 (2016).

  13. 13.

    Upton, J. W., Kaiser, W. J. & Mocarski, E. S. DAI/ZBP1/DLM-1 complexes with RIP3 to mediate virus-induced programmed necrosis that is targeted by murine cytomegalovirus vIRA. Cell Host Microbe 11, 290–297 (2012).

  14. 14.

    Daniels, B. P. et al. The nucleotide sensor ZBP1 and kinase RIPK3 induce the enzyme IRG1 to promote an antiviral metabolic state in neurons. Immunity 50, 64–76 (2019).

  15. 15.

    Newton, K. et al. RIPK1 inhibits ZBP1-driven necroptosis during development. Nature 540, 129–133 (2016).

  16. 16.

    Lin, J. et al. RIPK1 counteracts ZBP1-mediated necroptosis to inhibit inflammation. Nature 540, 124–128 (2016).

  17. 17.

    Lehle, A. S. et al. Intestinal inflammation and dysregulated immunity in patients with inherited caspase-8 deficiency. Gastroenterology 156, 275–278 (2019).

  18. 18.

    Li, Y. et al. Human RIPK1 deficiency causes combined immunodeficiency and inflammatory bowel diseases. Proc. Natl Acad. Sci. USA 116, 970–975 (2019).

  19. 19.

    Cuchet-Lourenço, D. et al. Biallelic RIPK1 mutations in humans cause severe immunodeficiency, arthritis, and intestinal inflammation. Science 361, 810–813 (2018).

  20. 20.

    Uchiyama, Y. et al. Primary immunodeficiency with chronic enteropathy and developmental delay in a boy arising from a novel homozygous RIPK1 variant. J. Hum. Genet. 64, 955–960 (2019

  21. 21.

    Kaiser, W. J., Upton, J. W. & Mocarski, E. S. Receptor-interacting protein homotypic interaction motif-dependent control of NF-κB activation via the DNA-dependent activator of IFN regulatory factors. J. Immunol. 181, 6427–6434 (2008).

  22. 22.

    Rebsamen, M. et al. DAI/ZBP1 recruits RIP1 and RIP3 through RIP homotypic interaction motifs to activate NF-κB. EMBO Rep. 10, 916–922 (2009).

  23. 23.

    Dannappel, M. et al. RIPK1 maintains epithelial homeostasis by inhibiting apoptosis and necroptosis. Nature 513, 90–94 (2014).

  24. 24.

    Welz, P. S. et al. FADD prevents RIP3-mediated epithelial cell necrosis and chronic intestinal inflammation. Nature 477, 330–334 (2011).

  25. 25.

    Yang, D. et al. ZBP1 mediates interferon-induced necroptosis. Cell. Mol. Immunol. (2019).

  26. 26.

    Ahmad, S. et al. Breaching self-tolerance to Alu duplex RNA underlies MDA5-mediated inflammation. Cell 172, 797–810 (2018).

  27. 27.

    Mannion, N. M. et al. The RNA-editing enzyme ADAR1 controls innate immune responses to RNA. Cell Rep. 9, 1482–1494 (2014).

  28. 28.

    Bae, S. et al. Energetics of Z-DNA binding protein-mediated helicity reversals in DNA, RNA, and DNA–RNA duplexes. J. Phys. Chem. B 117, 13866–13871 (2013).

  29. 29.

    Etchin, J. et al. KPT-330 inhibitor of CRM1 (XPO1)-mediated nuclear export has selective anti-leukaemic activity in preclinical models of T-cell acute lymphoblastic leukaemia and acute myeloid leukaemia. Br. J. Haematol. 161, 117–127 (2013).

  30. 30.

    Hafner, M. et al. Keratin 14 Cre transgenic mice authenticate keratin 14 as an oocyte-expressed protein. Genesis 38, 176–181 (2004).

  31. 31.

    Tröder, S. E. et al. An optimized electroporation approach for efficient CRISPR/Cas9 genome editing in murine zygotes. PLoS ONE 13, e0196891 (2018).

  32. 32.

    Vlantis, K. et al. NEMO prevents RIP kinase 1-mediated epithelial cell death and chronic intestinal inflammation by NF-κB-dependent and -independent functions. Immunity 44, 553–567 (2016).

  33. 33.

    Adolph, T. E. et al. Paneth cells as a site of origin for intestinal inflammation. Nature. 503, 272–276 (2013).

  34. 34.

    Upton, J. W., Kaiser, W. J. & Mocarski, E. S. Virus inhibition of RIP3-dependent necrosis. Cell Host Microbe 7, 302–313 (2010).

  35. 35.

    Attig, J., Young, G. R., Stoye, J. P. & Kassiotis, G. Physiological and pathological transcriptional activation of endogenous retroelements assessed by RNA-sequencing of B lymphocytes. Front. Microbiol. 8, 2489 (2017).

  36. 36.

    Wheeler, T. J. & Eddy, S. R. nhmmer: DNA homology search with profile HMMs. Bioinformatics 29, 2487–2489 (2013).

  37. 37.

    Kim, D., Langmead, B. & Salzberg, S. L. HISAT: a fast spliced aligner with low memory requirements. Nat. Methods 12, 357–360 (2015).

  38. 38.

    Liao, Y., Smyth, G. K. & Shi, W. featureCounts: an efficient general purpose program for assigning sequence reads to genomic features. Bioinformatics 30, 923–930 (2014).

  39. 39.

    Love, M. I., Huber, W. & Anders, S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 15, 550 (2014).

  40. 40.

    Kokot, M., Dlugosz, M. & Deorowicz, S. KMC 3: counting and manipulating k-mer statistics. Bioinformatics 33, 2759–2761 (2017).

  41. 41.

    Hubley, R. et al. The Dfam database of repetitive DNA families. Nucleic Acids Res. 44, D81–D89 (2016).

  42. 42.

    Raudvere, U. et al. g:Profiler: a web server for functional enrichment analysis and conversions of gene lists (2019 update). Nucleic Acids Res. 47, W191–W198 (2019).

Download references

Acknowledgements

We thank E. Gareus, J. Kuth, B. Kühnel, E. Stade, C. Uthoff-Hachenberg and J. von Rhein for technical assistance; B. Zevnik and the CECAD Transgenic Core Unit for the generation of mutant ZBP1 knock-in mice; A. Schauss and the CECAD Imaging Facility for microscopy support; and A. Athanasiadis for valuable discussions. Research reported in this publication was supported by funding from the European Research Council (grant agreement no. 787826), the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation; projects SFB829 (project no. 73111208), SFB1218 (project no. 269925409), SFB1399 (project no. 413326622), SFB1403 (project no. 414786233), PA 1476/8-1 (project no. 411102043) and CECAD (project no. 390661388)) and the Federal Ministry of Education and Research (BMBF, e:med project InCa, grant no. 01ZX1901A) to M.P., and by the Francis Crick Institute (FC001099) and the Wellcome Trust (102898/B/13/Z) to G.K. J.L. and R.O.E. were supported by postdoctoral fellowships from the Alexander von Humboldt Foundation.

Author information

H.J., L.W., S.K., R.S. and M.P. conceived the study and designed the experiments. H.J., L.W., S.K., R.S., J.L., R.O.E., A.F., R.L. and G.R.Y. performed and analysed experiments. W.J.K., G.K. and M.P. supervised the experiments. H.J., L.W., S.K., R.S., G.K., W.J.K. and M.P. interpreted data and wrote the paper.

Correspondence to Manolis Pasparakis.

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Competing interests

M.P. received consulting and speaker fees from Genentech, GSK, Boehringer Ingelheim and Sanofi.

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Extended data figures and tables

Extended Data Fig. 1 Generation of mutant Zbp1 knock-in mice.

ae, Schematic depicting the Zbp1 mutant mice generated using CRISPR–Cas9-mediated gene targeting in C57BL/6N zygotes, as indicated. To generate Zbp1mZα2 mice, amino acids N122 and Y126 in Zα2 domain were substituted with D and A, respectively (a). For Zbp1mZα1–2 mice, both Zα1 (N46D and Y50A) and Zα2 (N122D and Y126A) were mutated (b). For Zbp1mR1 mice, the residues of the core RHIM motif starting at amino acid position 192 (IQIG) were replaced by alanines (c). Sequencing traces of the desired mutations are shown in heterozygous mice. d, Zbp1ΔZα mice were generated by deleting exon 2 and exon 3, which contain both Zα domains (d). A Zbp1 knockout mouse was generated by targeting both exon 2 and exon 3. An allele containing a 373-bp deletion in exon 2 and the following intron as well as an additional 4-bp deletion in exon 3 was chosen as the Zbp1-knockout allele (e). f, Immunoblot analysis of total lysates from lung fibroblasts of the indicated genotypes, stimulated with IFNγ (1,000 U ml−1) for 24 h. One representative out of two independent experiments shown (f). GAPDH was used as a loading control for immunoblot analysis. For gel source data, see Supplementary Fig. 1.

Extended Data Fig. 2 ZBP1 exhibits Zα-dependent and -independent functions in antiviral defence.

a, Cell death analysed by measuring LDH release in supernatants of lung fibroblasts of the indicated genotypes infected with influenza A virus (at an MOI of 1 or 5) for 24 h. Data show mean from technical triplicates (n = 3). b, Immunoblot analysis of total lysates from lung fibroblasts of the indicated genotypes infected with influenza A virus (MOI of 5) for 8 h, with the indicated antibodies. GAPDH was used as a loading control. ce, Viral titres in spleen (c), liver (d) or salivary gland (e) from mice of the indicated genotypes at 5 d (c, d) or 14 d (e) after intraperitoneal inoculation with 1 × 106 MCMV-M45(mutRHIM). Graphs show values from individual mice, as well as mean ± s.e.m. P values by two-sided nonparametric Mann–Whitney test. The dotted horizontal red line depicts the detection limit of the assay. Data shown are representative of two independent experiments. For gel source data, see Supplementary Fig. 1. Source Data

Extended Data Fig. 3 ZBP1 causes keratinocyte necroptosis and skin inflammation in RIPK1E-KO mice by Zα-dependent and -independent mechanisms.

a, Representative images of skin sections from 6–7-week-old mice of the indicated genotypes, immunostained with the indicated antibodies and DAPI (DNA stain). RIPK1E-KO, n ≥ 5 mice; RIPK1E-KO Zbp1mZα2/mZα2, n ≥ 3 mice; RIPK1E-KO Zbp1ΔZα/ΔZα, n ≥ 3 mice; RIPK1E-KO Zbp1mR1/mR1, n ≥ 3 mice. Scale bars, 50 μm. b, Quantitative reverse-transcription PCR analysis of the mRNA expression of the indicated cytokines and chemokines, in RNA isolated from total skin from 6–7-week-old mice of the indicated genotypes. Each dot represents an individual mouse. Data show mean ± s.e.m. P value by two-sided nonparametric Mann–Whitney test. Control mice include Ripk1fl/fl mice that do not express K14-Cre or Ripk1fl/WTK14-Cre mice, with WT or mutated Zbp1 alleles. ce, Representative images of mice of the indicated genotypes at the indicated age. RIPK1E-KO, n = 6 mice and RIPK1E-KO Zbp1ΔZα/ΔZα, n = 17 mice (c); RIPK1E-KO Zbp1mZα2/mZα2, n = 16 mice (d); RIPK1E-KO Zbp1mR1/mR1, n = 20 mice (e). Source Data

Extended Data Fig. 4 ZBP1-mediated perinatal lethality in Ripk1mR/mR mice depends on Zα-dependent sensing of Z-NAs and RHIM1-mediated signalling.

a, Representative images of skin sections from mice of the indicated genotypes stained with H & E or immunostained with antibodies against keratin 10 and keratin 14 and counterstained with DAPI to visualize nuclear DNA. Skin sections from Ripk1mR/mR or Ripk1mR/WT embryos at embryonic day (E)18.5 are compared with skin from newborn (postnatal day (P)1 to P3) double-mutant mice. Scale bars, 100 μm. Ripk1mR/mR, n = 3 mice; Ripk1mR/WT, n = 3 mice; Ripk1mR/mR Zbp1ΔZα/ΔZα, n = 3 mice; Ripk1mR/mR Zbp1mZα2/mZα2, n = 3 mice; Ripk1mR/mR Zbp1mR1/mR1, n = 6 mice; Ripk1mR/WT Zbp1mR1/mR1, n = 3 mice. b, c, Immunoblot analysis of total lysates from primary lung fibroblasts (b) and keratinocytes (c) of the indicated genotypes stimulated with IFNγ (1,000 U ml−1) for 24 h. d, Spleen-to-body weight ratio of mice of the indicated genotypes. Each dot represents an individual mouse. Data show mean ± s.e.m. P value by two-sided unpaired t-test or nonparametric Mann–Whitney test. e, Representative H & E-stained sections from the spleen and liver of mice with the indicated genotypes. Ripk1WT/WT Zbp1−/WT, n = 5 mice; Ripk1mR/mR Zbp1−/−, n = 6 mice; Ripk1mR/mR Zbp1−/− Trif −/−, n = 6 mice; Ripk1mR/mR Zbp1ΔZα/ΔZα, n = 13 mice; Ripk1mR/WT Zbp1ΔZα/ΔZα, n = 10 mice; Ripk1mR/mR Zbp1mZα2/mZα2, n = 21 mice; Ripk1mR/WT Zbp1mZα2/mZα2, n = 3 mice; Ripk1mR/mR Zbp1mR1/mR1, n = 4 mice; Ripk1mR/WT Zbp1mR1/mR1, n = 6 mice. Representative data of two experiments are shown in b, c. GAPDH was used as a loading control for immunoblot analysis. For gel source data, see Supplementary Fig. 1. Source Data

Extended Data Fig. 5 IFNα and IFNγ induce the death of Ripk1mR/mR cells by inducing Zα-dependent ZBP1 activation and RHIM1-mediated downstream signalling.

a, Cell death measured by YOYO-1 uptake in Ripk1WT/WT or Ripk1mR/mR MEFs treated with IFNγ (1,000 U ml−1) for 48 h and IncuCyte images of Ripk1WT/WT or Ripk1mR/mR MEFs before and 48 h after IFNγ (1,000 U ml−1) treatment. YOYO-1 staining is shown in green. bf, h, Graphs depicting cell death assessment by YOYO-1 uptake in MEFs of the indicated genotypes treated with combinations of IFNγ (1,000 U ml−1), IFNα (50 ng ml−1), Z-VAD-FMK (20 μM) and GSK'872 (3 μM) for 48 h. g, Immunoblot analysis of total lysates from MEFs of the indicated genotypes stimulated with IFNγ (1,000 U ml−1) for 24 h. Representative data of two (g), three (c, d, f, h), five (b) or seven (a, e) experiments. Data in af, h are mean values from technical triplicates (n = 3). GAPDH was used as a loading control for immunoblot analysis. For gel source data, see Supplementary Fig. 1. Source Data

Extended Data Fig. 6 Treatment with IFNγ and emricasan in the presence of etanercept induces ZBP1-dependent cell death.

a, b, Graphs depicting cell death assessment by YOYO-1 uptake in wild-type and ZBP1-deficient MEFs treated with combinations of IFNγ (1,000 U ml−1, 24-h pretreatment), emricasan (em) (5 μM) and etanercept (et) (50 μg ml−1). c, Graph depicting cell death assessment by YOYO-1 uptake in wild-type MEFs treated with combinations of TNF (20 ng ml−1), birinapant (1 μM), Z-VAD-FMK (20 μM) and etanercept (50 μg ml−1). d, f, Graphs depicting cell death assessment by YOYO-1 uptake in MEFs (d) and lung fibroblasts (f) of the indicated genotypes treated with combinations of IFNγ (1,000 U ml−1) (24-h pretreatment), etanercept (50 μg ml−1) and emricasan (5 μM). e, Immunoblot analysis of total lysates from MEFs of the indicated genotypes stimulated with combinations of IFNγ (1,000 U ml−1) (24-h pretreatment), etanercept (50 μg ml−1) and emricasan (5 μM). Total lysate from wild-type MEFs treated with a combination of TNF (20 ng ml−1), birinapant (1 μM) and Z-VAD-FMK (20 μM) (TSZ) for 4 h was used as positive control for the detection of pMLKL and pRIPK3. g, Immunoblot analysis of total lysates from immortalized MEFs transduced with lentiviruses expressing Flag, Flag-tagged ZBP1 or Flag-tagged ZBP1(mZα1–2) stimulated with doxycycline (1 μg ml−1) for 24 h. Representative data of two (g), three (c, e), four (f), six (a, d) or nine (b) experiments. Data in ad, f are mean values from technical triplicates (n = 3). Data shown in f serve as control for the data shown in Fig. 2d, and come from the same experiment. GAPDH was used as a loading control for immunoblot analysis. For gel source data, see Supplementary Fig. 1. Source Data

Extended Data Fig. 7 RIPK1 deficiency in keratinocytes causes extensive transcriptional changes in the skin.

a, Differential expression (≥2-fold, P ≤ 0.05) of 4,204 genes in skin biopsies from 6-week-old wild-type (n = 5) and RIPK1E-KO mice (n = 5). b, Functional annotation by gene ontology (GO) of the top 1,000 upregulated genes and the top 1,000 downregulated genes between the samples in a, performed using g:Profiler. P values were estimated by hypergeometric distribution tests and adjusted by multiple-testing correction using the g:SCS (set counts and sizes) algorithm, integral to the g:Profiler server.

Extended Data Fig. 8 Treatment with reverse transcriptase inhibitors ameliorates skin inflammation in RIPK1E-KO mice.

a, Kaplan–Meier plot depicting lesion-free survival (top) and microscopy quantification of mean epidermal thickness and inflamed skin area (bottom) of mice of the indicated genotypes that were treated or not with reverse transcriptase inhibitors (RTi) from birth until the age of six weeks. Each dot represents an individual mouse. Data are mean ± s.e.m. P value by two-sided nonparametric Mann–Whitney test. The data from untreated control and RIPK1E-KO mice are included also in Figure 1b, c.  b, Hierarchical clustering of samples from control (n = 5) and RIPK1E-KO mice according to the expression of genes that are differentially expressed (≥1.75-fold, P ≤ 0.05, two-sided t-test, performed in Qlucore Omics Explorer 3.3) between untreated RIPK1E-KO mice (n = 5) and RIPK1E-KO mice treated with reverse transcriptase inhibitors (n = 5). Each column corresponds to one mouse. The mean epidermal thickness and inflammation score for each mouse are indicated. Control mice include Ripk1fl/fl mice that do not express K14-Cre or Ripk1fl/WTK14-cre mice. Source Data

Extended Data Fig. 9 Inhibition of nuclear export triggers Zα-dependent ZBP1-mediated cell death.

a, Immunoblot analysis of wild-type MEFs treated with TNF (20 ng ml−1), IFNα (50 ng ml−1), IFNβ (50 ng ml−1) or IFNγ (1,000 U ml−1) for 24 h. b, Cell death measured by YOYO-1 uptake in wild-type MEFs treated with combinations of IFNγ (1,000 U ml−1) (24-h pretreatment) and LMB (1 ng ml−1). IncuCyte images of wild-type MEFs before and after treatment with LMB for 24 h. YOYO-1 staining is shown in green. ce, Graphs depicting cell death assessment by YOYO-1 uptake in MEFs of the indicated genotypes treated with combinations of IFNγ (1,000 U ml−1) (24-h pretreatment), IFNα (50 ng ml−1) (24-h pretreatment) and LMB (1 ng ml−1). f, g, i, Graphs depicting cell death assessment by YOYO-1 uptake in lung fibroblasts of the indicated genotypes treated with combinations of IFNγ (1,000 U ml−1) (24-h pretreatment), KPT-330 (1  μM or 10  μM) (f) and LMB (5 ng ml−1) (g, i). h, Graphs depicting cell death assessment by YOYO-1 uptake in Mlkl−/− MEFs treated with combinations of IFNγ (1,000 U ml−1) (24-h pretreatment), LMB (1 ng ml−1) and emricasan (5 μM). j, Immunoblot analysis of total lysates from immortalized MEFs transduced with lentiviruses expressing Flag, Flag-tagged ZBP1 or Flag-tagged ZBP1(mZα1–2) stimulated with combinations of doxycycline (1 μg ml−1) (24-h pretreatment) and LMB (5 ng ml−1). k, Graphs depicting cell death assessment by YOYO-1 uptake in immortalized MEFs transduced with lentiviruses expressing Flag, Flag-tagged ZBP1 or Flag-tagged ZBP1(mZα1–2) stimulated with combinations of doxycycline (1 μg ml−1) (24-h pretreatment) and LMB (5 ng ml−1). Dox, doxycycline. Representative data of 2 (a, j), 3 (k), 4 (fh), 5 (i) or 6 (be) experiments. Data in bi, k are mean values from technical triplicates (n = 3). Data shown in c, i serve as controls for the data shown in Fig. 4a, e, respectively, and come from the same experiments. GAPDH was used as a loading control for immunoblot analysis. For gel source data, see Supplementary Fig. 1. Source Data

Extended Data Fig. 10 Schematic depicting the regulation of ZBP1-mediated activation of RIPK3–MLKL-dependent necroptosis by RIPK1 and caspase-8.

The sensing of endogenous cellular Z-RNA by its Zα domains activates ZBP1, inducing its interaction with RIPK3; however, cell death is inhibited owing to negative regulation by RIPK1 and caspase-8. RIPK1 inhibits the ZBP1-induced activation of RIPK3 by FADD-mediated recruitment of caspase-8, which cleaves components of the complex such as RIPK1 and RIPK3. In cells that lack RIPK1 or express RIPK1 with a mutated RHIM, in FADD-deficient cells, and in cells treated with caspase inhibitors, the Zα-dependent sensing of endogenous Z-RNA activates ZBP1, which strongly engages RIPK3 and then triggers MLKL-dependent necroptosis.

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Supplementary Information Fig. 1: Uncropped scans of immunoblots presented in the manuscript.

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Supplementary Data

Supplementary Data File 1: Gene transfer format (GTF) file of repetitive region annotations for the mouse genome (GRCm38.78), with the adjacent annotations for the same element merged.

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Supplementary Data File 2: Specific shell commands executed and Python code to convert kmers into complementary kmers.

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Jiao, H., Wachsmuth, L., Kumari, S. et al. Z-nucleic-acid sensing triggers ZBP1-dependent necroptosis and inflammation. Nature (2020). https://doi.org/10.1038/s41586-020-2129-8

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